Methods for fixing tissue samples using aqueous formaldehyde solutions have been known for some time.
A saturated aqueous formaldehyde solution having 37 percent formaldehyde by weight, or 40 percent formaldehyde by volume, is also referred to as “formalin.” Indications as to the concentration of formaldehyde solutions that are used for fixing tissue samples typically refer to this. For example, a “ten-percent formalin” solution contains 4 percent formaldehyde by volume.
In aqueous solution formaldehyde quickly hydrates to methylene glycol, which in turn reacts with macromolecules such as proteins and glycoproteins in the tissue being fixed. What occurs firstly is the formation, on suitable amine, amide, and reactive alcohol groups among others, of hydroxymethyl groups. Only with longer fixing times do these become crosslinked to an appreciable extent.
Formaldehyde also reacts with proteins in the cell nucleus, thereby stabilizing the protein sheath of the nucleic acids. The free amino groups of the nucleic acids themselves can also react with formaldehyde, as can double bonds and thiol groups in unsaturated fatty acids. Pure carbohydrates, on the other hand, generally do not react with formaldehyde.
The reactive groups of peptides and proteins that react most intensively with methylene glycol, and thus respond best to formaldehyde fixing, are the amine groups of lysine, cysteine, histidine, arginine, and tyrosine, and the hydroxyl groups of serine and threonine. So-called “over-fixing” (tanning) results in crosslinking of lysine and of the amides of the protein backbone. Such reactions seldom occur because of the shorter fixing times typically used nowadays.
Formaldehyde has good fixing properties, but is highly hazardous to health and, upon exposure, can cause allergies and irritation of the skin, respiratory tract, and eyes. Formaldehyde is furthermore regarded as carcinogenic. Efforts are therefore being made to replace formaldehyde with less harmful compounds for the fixing of tissue samples. The same is also true of other fixing agents such as glutaraldehyde and osmium tetroxide.
In this connection it is possible to use compounds that release suitable aldehydes, especially formaldehyde. One example thereof is urotropin. The use of such compounds is known, for example, from EP 1 895 287 B1 (DE 10 2006 040 315 B4, U.S. Pat. No. 7,915,007 B2). They are also referred to as “releasers” or “donors” of the respective aldehydes.
The use of aldehyde-releasing compounds is disadvantageous in that in an equilibrium reaction they normally release only that quantity of aldehyde which is consumed by the reaction with the tissue sample. The fixing speed is therefore relatively slow as compared with conventional formalin solutions in which formaldehyde, or its hydration product methylene glycol, is present to excess. Fixing methods using corresponding donors therefore present disadvantages despite the simpler utilization and the reduced health hazard.
A need therefore exists for improved, in particular accelerated capabilities for fixing tissue samples using compounds that release aldehydes.